Antagonists of HMG1 for treating inflammatory conditions

ABSTRACT

There is disclosed a pharmaceutical composition and method for treating sepsis, including septic shock and ARDS (acute respiratory distress syndrome), comprising administering an effective amount of a HMG1 antagonist. There is further disclosed a diagnostic method for monitoring the severity or potential lethality of sepsis or septic shock, comprising measuring the serum concentration of HMG1 in a patient exhibiting or at risk or exhibit sepsis or septic shock symptoms. Lastly, there is disclosed a pharmaceutical composition and method for effecting weight loss or treating obesity, comprising administering an effective amount of HMG1 or a therapeutically active HMG1 fragment.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/210,747, filed Jul. 31, 2002, which is a continuation of U.S.application Ser. No. 09/503,632, filed Feb. 14, 2000, now issued as U.S.Pat. No. 6,468,533, which is a divisional of U.S. application Ser. No.09/248,574, filed Feb. 11, 1999, now issued as U.S. Pat. No. 6,303,321.The entire teachings of the above applications are incorporated hereinby reference.

TECHNICAL FIELD OF THE INVENTION

The present invention provides a pharmaceutical composition and methodfor treating diseases characterized by activation of an inflammatorycytokine cascade, particularly sepsis, including septic shock and ARDS(acute respiratory distress syndrome), comprising administering aneffective amount of an antagonist to the high mobility group 1 protein(HMG1). The present invention further provides a diagnostic method formonitoring the severity of sepsis and related conditions, comprisingmeasuring the serum concentration of HMG1 in a patient exhibitingsymptoms of a disease characterized by activation of inflammatorycytokine cascade. Lastly, the present invention provides apharmaceutical composition and method for effecting weight loss ortreating obesity, comprising administering an effective amount of anHMG1 protein or a therapeutically active fragment of the gene product ofan HMG1 gene.

BACKGROUND OF THE INVENTION

Sepsis is an often fatal clinical syndrome that develops after infectionor injury. Sepsis is the most frequent cause of mortality inhospitalized patients. Experimental models of gram negative sepsis basedon administration of bacterial endotoxin (lipopolysaccharide, LPS) haveled to an improved understanding of the pathogenic mechanisms of lethalsepsis and conditions related to sepsis by virtue of the activation of acommon underlying inflammatory cytokine cascade. This cascade ofhost-response mediators includes TNF, IL-1, PAF and othermacrophage-derived factors that have been widely studied as acute, earlymediators of eventual lethality in severe endotoxemia (Zhang and Tracey,In The Cytokine Handbook, 3rd ed. Ed. Thompson (Academic Press Limited,USA). 515–547,1998).

Unfortunately, therapeutic approaches based on inhibiting theseindividual “early” mediators of endotoxemia have met with only limitedsuccess in large prospective clinical trials against sepsis in humanpatients. It is possible to infer from these disappointing results thatlater-appearing factors in the host response might critically determinepathogenesis and/or lethality in sepsis and related disorders.Accordingly, there is a need to discover such putative “late” mediatorsnecessary and/or sufficient for part or all of the extensive multisystempathogenesis, or for the lethality, of severe endotoxemia, particularlyas endotoxemia is representative of clinical sepsis and related clinicaldisorders.

HMG1 is a 30 kDa chromosomal nucleoprotein belonging to the burgeoninghigh mobility group (HMG) of non-histone chromatin-associated proteins.As a group, the HMG proteins recognize unique DNA structures and havebeen implicated in diverse cellular functions, including determinationof nucleosome structure and stability, as well as in transcriptionand/or replication. The HMG proteins were first characterized by Johnsand Goodwin as chromatin components with a high electrophoretic mobilityin polyacrylamide gels (see in The HMG Chromosomal Proteins, E. W.Johns, Academic Press, London, 1982). Higher eukaryotes exhibit threefamilies of HMG proteins: the HMG-1/-2 family, the HMG-14/-17 family andthe HMG-I/-Y family. Although the families are distinguishable by sizeand DNA-binding properties, they are similar in their physicalproperties. HMG proteins are highly conserved across species,ubiquitously distributed and highly abundant, and are extractable fromchromatin in 0.35 M NaCl and are soluble in 5% perchloric ortrichlioroacetic acid. Generally, HMG proteins are thought to bend DNAand facilitate binding of various transcription factors to their cognatesequences, including for instance, progesterone receptor, estrogenreceptor, HOX proteins, and Oct1, Oct2 and Oct6. Recently, it has becomeapparent that a large, highly diverse group of proteins includingseveral transcription factors and other DNA-interacting proteins,contain one or more regions similar to HMG1, and this feature has cometo be known as the HMG1 box or HMG1 domain. cDNAs coding for HMG1 havebeen cloned from human, rat, trout, hamster, pig and calf cells, andHMG1 is believed to be abundant in all vertebrate cell nuclei. Theprotein is highly conserved with interspecies sequence identities in the80% range. In chromatin, HMG1 binds to linker DNA between nucleosomesand to a variety of non-β-DNA structures such as palindromes, cruciformsand stem-loop structures, as well as cisplatin-modified DNA. DNA bindingby HMG1 is generally believed to be sequence insensitive. HMG1 is mostfrequently prepared from washed nuclei or chromatin, but the protein hasalso been detected in the cytoplasm. (Reviewed in Landsman and Bustin,BioEssays 15:539–546, 1993; Baxevanis and Landsman, Nucleic AcidsResearch 23:514–523, 1995). To date, no link has been establishedbetween the HMG proteins and any clinical condition or disease.

HMG1 has been alternatively identified as a heparin-binding proteinabundantly expressed in developing brain and dubbed “amphoterin” for itshighly dipolar sequence, comprising two internal repeats of a positivelycharged domain of about 80 amino acids (the HMG1 box) and an acidicC-terminal domain containing a stretch of approximately 30 continuousglutamic or aspartic acid residues. Amphoterin/HMG1 has been localizedto the outer surface of the plasma membranes of epithelial, andespecially neuronal cells, where it has been specifically localized tothe filipodia of neural cells. Inhibition studies have suggested thatamphoterin/HMG1 is required for process (neurite) extension andamphoterin/HGM1 also may be involved in neuron-glia interactions(Meremnies et al., J. Biol. Chem. 266:16722–16729,1991; Merenmies etal., J Biol. Chem. 266:16722–16729, 1991; Milev et al., J Biol. Chem.273:6998–7005, 1998; and Salmivirta et al., Exp. Cell Res. 200:444–451,1992). Amphotelin/HMG1 can be released from murine erythroleukemia cellsafter stimulation with the chemical inducer hexamethylenebisacetamide(Melloni et al., Biochem. Biophys. Res. Commun. 210:82–89, 1995).Previous study suggested that the gene product of the HMG1 genefunctions as a differentiation enhancing factor by stimulating α-PKC(Melloni et al., Biochem. Biophys. Res. Commun. 210:82–89, 1995; andMelloni et al., FEBS Lett. 368:466–470, 1995).

The HMG1 gene product has been shown to interact with plasminogen andtissue-type plasminogen activator (t-PA) and effectively enhance plasmingeneration at the cell surface, a system that is known to play a role inextracellular proteolysis during cell invasion and tissue remodeling.Amphoterin/HMG1 has also been shown to interact with the receptor ofadvanced glycosylation end products (RAGE) (Mohan et al., Biochem.Biophys. Res. Commun. 182:689–696, 1992; Yamawaki et al., J. Neurosci.Res. 44:586–593, 1996; Salmivirta et al., Exp. Cell Res. 200:444–451,1992; and Vassalli et al., J. Clin. Invest. 88:1067–1072, 1991),(Redlitz and Plow, Baillieres Clin. Haematol. 8:313–327, 1995; andParkkinen et al., J. Biol. Chem. 266:16730–16735, 1991).

There is a longstanding need in the art to discover improved agents thatcan prevent the cytokine-mediated inflammatory cascade and havetherapeutic activity in a large variety of cytokine-mediatedinflammatory diseases. The present invention was made during the courseof investigative research to identify agents that mediate toxicity,pathogenesis and/or lethality in sepsis and other disorders related by acommon activation of the inflammatory cytokine cascade.

Diseases and conditions mediated by the inflammatory cytokine cascadeare numerous. Such conditions include the following grouped in diseasecategories:

-   -   Systemic Inflammatory Response Syndrome, which includes:        -   Sepsis syndrome            -   Gram positive sepsis            -   Gram negative sepsis            -   Culture negative sepsis            -   Fungal sepsis            -   Neutropenic fever            -   Urosepsis        -   Meningococcemia        -   Trauma hemorrhage        -   Hums        -   Ionizing radiation exposure        -   Acute pancreatitis        -   Adult respiratory distress syndrome (ARDS)    -   Reperfusion Injury, which includes        -   Post-pump syndrome        -   Ischemia-reperfusion injury    -   Cardiovascular Disease, which includes        -   Cardiac stun syndrome        -   Myocardial infarction        -   Congestive heart failure    -   Infectious Disease, which includes        -   HIV infection/HIV neuropathy        -   Meningitis        -   Hepatitis        -   Septic arthritis        -   Peritonitis        -   Pneumonia Epiglottitis        -   E. coli 0157:H7    -   Hemolytic uremic syndrome/thrombolytic tbrombocytopcnic purpura    -   Malaria    -   Dengue hemorrhagic fever    -   Leishmaniasis    -   Leprosy    -   Toxic shock syndrome    -   Streptococcal myositis    -   Gas gangrene        -   Mycobacterium tuberculosis        -   Mycobaclerium aviun intracellulare        -   Pneumocystis carinii pneumonia        -   Pelvic inflammatory disease        -   Orchitis/epidydimitis        -   Legionella        -   Lyme disease        -   Influenza A        -   Epstein-Barr Virus        -   Viral associated hemiaphagocytic syndrome        -   Viral encephalitis/aseptic meningitis    -   Obstetrics/Gynecology, including:        -   Premature labor        -   Miscarriage        -   Infertility    -   Inflammatory Disease/Autoimmunity, which includes:        -   Rheumatoid arthritis/seronegative arthropathies        -   Osteoarthritis        -   Inflammatory bowel disease        -   Systemic lupus erythematosis        -   Iridoeyelitis/uveitistoptic neuritis        -   Idiopathic pulmonary fibrosis        -   Systemic vasculitis/Wegener's gramilornatosis        -   Sarcoidosis        -   Orchitis/vasectomy reversal procedures    -   Allergic/Atopic Diseases, which includes:        -   Asthma        -   Allergic rhinitis        -   Eczema        -   Allergic contact dermatitis        -   Allergic conjunctivitis        -   Hypersensitivity pneumonitis    -   Malignancy, which includes:        -   ALL        -   AML        -   CML        -   CLL        -   Hodgkin's disease, non-Hodgkin's lymphoma        -   Kaposi's sarcoma        -   Colorectal carcinoma        -   Nasopharyngeal carcinoma        -   Malignant histiocytosis        -   Paraneoplastic syndrome/hypercalcemia of malignancy    -   Transplants, including:        -   Organ transplant rejection        -   Graft-versus-host disease    -   Cachexia    -   Congenital, which includes:        -   Cystic fibrosis            -   Familial hematophagocytic lymphohistiocytosis            -   Sickle cell anemia    -   Dermatologic, which includes:        -   Psoriasis        -   Alopecia    -   Neurologic, which includes:        -   Multiple sclerosis        -   Migraine headache    -   Renal, which includes:        -   Nephrotic syndrome        -   Hemodialysis        -   Uremia    -   Toxicity, which includes:        -   OKT3 therapy        -   Anti-CD3 therapy        -   Cytokine therapy        -   Chemotherapy        -   Radiation therapy        -   Chronic salicylate intoxication    -   Metabolic/Idiopathic, which includes:        -   Wilson's disease        -   Hemachromatosis        -   Alpha-1 antitrypsin deficiency        -   Diabetes        -   Hashimoto's thyroiditis        -   Osteoporosis        -   Hypothalamic-pituitary-adrenal axis evaluation        -   Primary biliary cirrhosis

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition for treatingconditions (diseases) mediated by the inflammatory cytokine cascade,comprising an effective amount of an antagonist or inhibitor of HMG1.Preferably, the HMG1 antagonist is selected from the group consisting ofantibodies that bind to an HMG1 protein, HMG1 gene antisense sequencesand HMG1 receptor antagonists. The present invention provides a methodfor treating a condition mediated by the inflammatory cytokine cascade,comprising administering an effective amount of an HMG1 antagonist. Inanother embodiment, the inventive method further comprises administeringa second agent in combination with the HMG1 antagonist, wherein thesecond agent is an antagonist of an early sepsis mediator, such as TNF,IL-1α, IL-1β, MIF or IL-6. Most preferably, the second agent is anantibody to TNF or an IL-1 receptor antagonist (IL-1ra).

The present invention further provides a diagnostic and prognosticmethod for monitoring the seventy and predicting the likely clinicalcourse of sepsis and related conditions for a patient exhibitingshock-like symptoms or at risk to exhibit symptoms associated withconditions mediated by the inflammatory cascade. The inventivediagnostic and prognostic method comprises measuring the concentrationof HMG1 in a sample, preferably a serum sample, and comparing thatconcentration to a standard for HMG1 representative of a normalconcentration range of HMG1 in a like sample, whereby higher levels ofHMG1 are indicative of poor prognosis or the likelihood of toxicreactions. The diagnostic method may also be applied to other tissue orfluid compartments such as cerebrospinal fluid or urine. Lastly, thepresent invention provides a pharmaceutical composition and method foreffecting weight loss or treating obesity, comprising administering aneffective amount of HMG1 or a therapeutically active fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two graphs that profile the induction of HMG1 release byLPS in vitro (FIG. 1A) and in vivo (FIG. 1B). Specifically, FIG. 1Ashows the accumulation of HMG1 in culture supernatants of macrophage RAW264.7 cells after stimulation with LPS (100 ng/ml). The inset is aWestern blot (using antibodies raised against recombinant HMG1) showinginduction of HMG1 release from RAW 264.7 cells after induction with TNF.FIG. 1B shows accumulation of HMG1 in serum of LPS-treated mice. Serumfrom Balb/C mice was collected at various time points after LPSadministration, and assayed for HMG1 by Western blotting usingantibodies raised against recombinant HMG1.

FIG. 2 illustrates that HMG1 is a mediator of pathogenesis and lethalityin endotoxemia. FIG. 2A shows the protective effect of anti-HMG1antibodies against LPS lethality, tested in mice. Administration ofanti-HMG1 antiserum in the indicated amounts at −0.5 (if one dose), −0.5and 12 (if two doses), or −0.5, 12 and 36 (if three doses) hoursrelative to LPS challenge (at time 0) was protective against LPS-inducedlethality, and repeated dosing schedules provided better protection.FIG. 2B illustrates that rHMG1 caused dose-dependent lethality inendotoxic mice. Male Balb/C mice (20–23 grams) were randomized in groupsof ten to receive LPS (3.15 mg/kg; a non-lethal dose) alone or incombination with purified recombinant HMG1 protein. Administration ofHMG1 at the indicated doses 2, 16, 28 and 40 hours after LPS challengesignificantly increased the lethality of the underlying endotoxemia.FIG. 2C illustrates independent lethal toxicity of HMG1 as a function ofdose. Purified rHMG1 was administered to male Balb/C mice (five mice pertreatment group) as a single i.p. bolus at the indicated dosage. Micewere observed for at least 48 hours, and 60% of mice treated with rHMG1at a dose of 500 μg/mouse died within 24 hours of rHMG1 challenge,indicating a single dose LD₅₀ of less than 500 μg/mouse.

FIG. 3 shows that HMG1 induced TNF release both in vitro (FIG. 3A) andin vivo (FIG. 3B). Specifically, FIG. 3A shows that HMG1 induces TNFrelease from huPBMCs in dose-dependent fashion. Freshly isolated huPBMCcultures were stimulated with purified recombinant HMG1 protein at theindicated doses, and culture media were sampled four hours later to beassayed for TNF according to known immunologic methods (ELISA). FIG. 3Ashows the mean ±S.E.M. of the induced TNP response in two experiments(in triplicate). FIG. 3B shows that administration of HMG1 inducedaccumulation of TNF in serum of treated mice. Balb/C mice (20–23 g) weretreated intraperitoneally with purified recombinant HMG1 at theindicated doses and blood samples were taken two hours later for assayof TNF by an L929 bioassay and (TNF levels expressed as mean ±S.E.M.,N=3).

FIG. 4 shows that HMG1 caused body weight loss in mice. Purified HMG1was administered intraperitoneally to mice at 100 μg/mouse/day for threedays, and body weight was monitored. FIG. 4 shows the mean ±S.E.M. ofnet body weight change of three mice per group.

FIG. 5 shows the tissue distribution of HMG1 mRNA. Human RNA masterblots containing poly(A)⁺ RNA of various tissues (Clontech, Palo Alto,Calif. USA) were hybridized with a 0.6 kb digoxigenin-11-dUTP-labeledHMG1 cDNA probe synthesized by PCR using recombinant plasmid containingthe HMG1 cDNA insert, all in accordance with methods well-known in theart. Briefly, hybridization was performed in a hybridization buffer(5×SSC/2% blocking reagent/0.1% SDS/50% formamide, Boehringer Mannheim,Indianapolis, Ind.) with a probe concentration of 10 ng/ml for 16 hoursat 65° C. After hybridization, the filter was subjected to two washes of0.5×SSC/0.1% SDS for 5 minutes, and two washes of 0.2×SSC/0.1% SDS for10 minutes at room temperature. Signal was detected usinganti-digoxigenin antibodies conjugated to phosphotase and detectionreagents 4-nitrobluetetrazolium chloride (NBT) and5-cromo-4-chloro-3-indolyl-phosphate (BCIP) (Boehringer-Manuheim)according to standard methods. The blots were scanned with a silverimage scanner (Silverscanner II, Lacie Limited, Beaverton, Oreg.), andrelative optical density (in arbitrary units, AU) was quantified usingNIH 1.59 image software. Note that highest levels were observed inmacrophage-rich tissues.

FIG. 6 shows, in comparison to a group of normal control subjects,increased human serum HMG1 levels as detected in hospitalized humansubjects with sepsis, wherein the septic patients have been furthercategorized as to whether the patient died or survived.

FIG. 7 is a graph of the effect of 0 μg/ml, 5 μg/ml, 10 μg/ml, or 25μg/ml of HMG1 A box protein on the release of TNF (as a percent of HMG1mediated TNF release alone) from RAW 264.7 cells.

FIG. 8 is a histogram of the effect of HMG1 (0 or 1.5 μg/ml), HMG1 A box(0 or 10 μg/ml), or vector (0 or 10 μg/ml), alone, or in combination onthe release of TNF (as a percent of HMG1 mediated TNT release alone)from RAW 264.7 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery and isolation of ahighly inducible 30 kDa protein that is released by, and accumulates inmedia conditioned by, cultured murine macrophage-like cells (RAW 264.7)following stimulation with LPS, TNF, or IL-1. A partial amino acidsequence of this isolated polypeptide was identical to the sequence ofthe HMG1 protein, also known as amphoterin, a protein not before linkedto the pathogenesis of any disease. This information was used to clone acDNA encoding HMG1, which sequence was expressed to provide recombinantprotein, which protein was used to generate specific anti-HMG1antibodies.

Therapeutic and diagnostic efficacy was determined in a series ofpredictive in vitro and in vivo experiments. The experiments aredetailed in the Examples section. For example, following administrationof endotoxin (LD₁₀₀) to mice, serum HMG1 levels increased later (at 16h) than well-known “early” mediators of sepsis (such as TNF and IL-1)and plateau levels of HMG1 were maintained for 16 to 32 hours. Patientswith lethal sepsis had high serum HMG1 levels, which were not detectedin normal healthy volunteers. Moreover, acute experimentaladministration of rHMG1 to test animals, whether alone or in combinationwith sub-lethal amounts of LPS, caused marked pathological responses andeven death. More distributed dosing schedules of lower amounts of rHMG1led to significant weight loss in treated animals. These results giveevidence that HMG1 is a mediator of endotoxemia and particularly a latemediator, as opposed to known “early” mediators such as TNF and IL-1.These data further show the importance of serum HGM1 as a marker for theseverity or potential lethality of sepsis and related conditions.

In addition, treatment with anti-HMG1 antibodies provided fullprotection from LD₁₀₀ doses of LPS in mice. HMG1 is inducible by TNF andIL-1β, and dose-dependently stimulates TNF release from huPBMCs. TNF isa marker of macrophage activation, so it is likely (without limitationas to implied mechanisms or being bound by theory) that HMG1 promotesdownstream re-activation of cytokine cascades which, in turn, mediateslate pathogenesis and lethality in sepsis and related conditionsinvolving activation of pro-inflammatory cytokine responses. Thus, HMG1likely occupies a central role in mediating the inflammatory response toinfection and injury, and antagonists of HMG1 will be of therapeuticbenefit in sepsis and related conditions of inflammatory cascadeactivation. The appearance of HMG1 in the inflammatory cytokine cascadeis suitable to propagate later phases of the host response andcontribute to toxicity and lethality. The predictive data providedherein support the therapeutic efficacy of HMG1 antagonists and provideevidence in support of the aforementioned theory regarding mechanism ofaction. The in vivo treatment data showed the efficacy of HMG1antagonists in general, and anti-HMG1 antibodies in particular, fortreating conditions mediated by the inflammatory cytokine cascade ingeneral and particularly sepsis conditions, including, for example,septic shock, sepsis syndrome or other “sepsis-like” conditions mediatedby inflammatory cytokines. Further, the independent pathogenicity andtoxicity/lethality of HMG1 shows that HMG1 antagonists are particularlyeffective when co-administered with antagonists of “early” inflammatorymediators such as TNF, MIF, IL-1 and IL-6.

In summary, HMG1 is a cytokine mediator of inflammatory reactionsbecause: 1) HMG1 is released from macrophages and pituicytes followingstimulation with bacterial toxins or with pro-inflammatory cytokines(TNF or IL-1β), 2) HMG1 accumulates in serum of animals exposed to LPSand in patients with sepsis; and 3) HMG1-specific antibodies protectagainst mortality in a predictive lethal endotoxemia animal model ofclinical sepsis and related conditions.

Pharmaceutical Composition and Method of Administration

The inventive pharmaceutical composition or inventive pharmaceuticalcombination can be administered to a patient either by itself (complexor combination) or in pharmaceutical compositions where it is mixed withsuitable carriers and excipients. The inventive pharmaceuticalcomposition or inventive pharmaceutical combination can be administeredparenterally, such as by intravenous injection or infusion,intraperitoneal injection, subcutaneous injection, or intramuscularinjection. The inventive pharmaceutical composition or inventivepharmaceutical combination can be administered orally or rectallythrough appropriate formulation with carriers and excipients to formtablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like. The inventive pharmaceutical composition or inventivepharmaceutical combination can be administered topically, such as byskin patch, to achieve consistent systemic levels of active agent. Theinventive pharmaceutical composition or inventive pharmaceuticalcombination can be formulated into topical creams, skin or mucosalpatches, liquids or gels suitable for topical application to skin ormucosal membrane surfaces. The inventive pharmaceutical composition orinventive pharmaceutical combination can be administered by inhaler tothe respiratory tract for local or systemic treatment.

The dosage of the inventive pharmaceutical composition or inventivepharmaceutical combination of the present invention can be determined bythose skilled in the art from this disclosure. The pharmaceuticalcomposition or inventive pharmaceutical combination will contain aneffective dosage (depending upon the route of administration andpharmacokinetics of the active agent) of the inventive pharmaceuticalcomposition or inventive pharmaceutical combination and suitablepharmaceutical carriers and excipients, which are suitable for theparticular route of administration of the formulation (i.e., oral,parenteral, topical or by inhalation). The active agent is mixed intothe pharmaceutical formulation by means of mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping orlyophilizing processes. The pharmaceutical formulations for parenteraladministration include aqueous solutions of the active agent orcombination in water-soluble form. Additionally, suspensions of theactive agent may be prepared as oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may optionally containstabilizers or agents to increase the solubility of the active agent orcombination to allow for more concentrated solutions.

Pharmaceutical formulations for oral administration can be obtained bycombining the active agent with solid excipients, such as sugars (e.g.,lactose, sucrose, mannitol or sorbitol), cellulose preparations (e.g.,starch, methyl cellulose, hydroxypropylmethyl cellulose, and sodiumcarboxymethyl cellulose), gelaten, gums, or polyvinylpyrrolidone. Inaddition, a disintegrating agent may be added, and a stabilizer may beadded.

Antisense Oligomers

The present invention provides antisense oligomers having a sequenceeffective to inhibit or block the expression of the HMG1 gene or mRNAsequence. Anti sense technology, which uses specific-oligonucleotides toinhibit expression of target gene products, is developing as atherapeutic modality for human disease. Several selection criteria areavailable to contribute to the optimization of antisense oligonucleotideantagonists. For example, it is advisable to choose sequences with 50%or more GC content. Preferred sequences span the AUG initiation codon ofthe target protein, but sites in the coding region and 5′ UTR mayperform equally well. Such sequences are generally about 18–30nucleotides long and chosen to overlap the ATG initiation codon from theHMG1 cDNA sequence to inhibit protein expression. Longer oligomers areoften found to inhibit the target to a greater extent, indicating that apreferred length is about 25 mer for the first oligonucleotides chosenas antisense reagents. Typically, three oligonucleotide sequences arechosen with regard to these criteria, and compared for antagonistactivity to control oligonucleotide sequences, such as “reverse”oligonucleotides or those in which about every fourth base of theantisense sequence is randomized. Therefore, a preferred sequence formaking antisense oligomer sequences to HMG1 is a 25 mer sequence chosento overlap the ATG initiation codon (underlined) from the HMG1 cDNAsequence:

[SEQ ID NO. 5] GAGGAAAAATAACTAAACATGGGCAAAGGAGATCCTAAGAAGand such preferred antisense sequences are used to construct antisenseoligonucleotide agents (and suitable controls) for an its vitrocomparison as antagonists of HMG1. These in vitro data are predictive ofhuman clinical utility using antisense agents of comparable design.HMG1-Directed Antibodies

The antibodies disclosed herein may be polyclonal or monoclonal; may befrom any of a number of human, non-human eukaryotic, cellular, fungal orbacterial sources; may be encoded by genomic or vector-borne codingsequences; and may be elicited against native or recombinant HMG1 orfragments thereof with or without the use of adjuvants, all according toa variety of methods and procedures well-known in the art for generatingand producing antibodies. Generally, neutralizing antibodies againstHMG1 (i.e., those that inhibit biological activities of HMG1particularly with regard to its pro-inflammatory cytokine-like role) arepreferred for therapeutic applications while non-neutralizing antibodiesmay be as suitable for diagnostic applications. Examples of such usefulantibodies include but are not limited to polyclonal, monoclonal,chimeric, single-chain, and various human or humanized types ofantibodies, as well as various fragments thereof such as Fab fragmentsand fragments produced from specialized expression systems.

Diagnostic Assay

The diagnostic assay provided here uses anti-HMG1 antibodies that can beeither polycolonal or monoclonal or both. The diagnostic procedure canutilize standard antibody-based techniques for measuring concentrationsof the gene product of HMG1 genes in a biological fluid. Preferredstandard diagnostic procedures are ELISA assays and Western techniques.

EXAMPLE 1 Identification of HMG1 as a “late” Mediator of Endotoxemia

This example provides the results of an experiment to identify andisolate later released macrophage-derived factors that play a role insepsis and in related conditions typified by inflammatory cytokineactivity. The experiment reported in this example examined murinemacrophage RAW 264.7 cell-conditioned media after stimulation of thecultures with TNF. Murine macrophage RAW 264.7 cells were obtained fromAmerican Type Culture Collections (ATCC, Rockville, Md. USA), andproliferated in culture under DMEM supplemented with 10% fetal bovineserum and 1% glutamine. When confluency reached 70–80%, the medium wasreplaced by serum-free OPTI-MEM I medium and cultures were stimulatedwith pro-inflammatory cytokines (e.g., TNFα or IL-1) or bacterialendotoxin (LPS).

The proteins released from the above stimulated macrophage cultures weresurveyed. Specifically, at different time points, cells andcell-conditioned media were separately collected by centrifugation (3000rpm, 10 minutes). Proteins in the conditioned mediam were concentratedby ultrafiltration over Amicon membranes with Mr cutoff of 10 kDa(Amicon Inc., Beverly, Mass., USA), subsequently fractionated bySDS-PAGE, and stained with Coomassie blue (1.25% Coomassie Blue R250 in30% methanol/10% acetic acid). After destaining with 30% methanol/7%acetic acid, protein(s) of interest (i.e., those that preferentiallyaccumulated in conditioned media of stimulated cultures) was isolated byexcision from the SDS-PAGE gel, and subjected to N-terminal sequencinganalysis (Commonwealth Biotechnologies, Inc., Richmond, Va., USA).

Comparison of SDS-PAGE gel analysis of profiles of proteins accumulatedin control (without TNFα stimulation) versus TNF-stimulated RAW 264.7cells revealed a strongly inducible 30 kDa protein whose concentrationin the cell-conditioned medium was significantly increased afterstimulation for 16 hours. Amino acid sequence analysis of this isolatedprotein revealed its N-terminal sequence asGly-Lys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys-Met-Ser-Ser [SEQ ID NO. 1].A review of relevant gene databases found a 100% identity to theN-terminal amino acid sequence of HMG1.

These data identified HMG1 as a “late-appearing” product ofLPS-stimulated macrophage cultures, and therefore as a candidatepro-inflammatory mediator. This activity was confirmed by administrationof recombinantly produced HMG1 and/or of anti-HMG1 antibodies incellular and animal model systems that are predictive of human clinicalconditions.

EXAMPLE 2 Cellular Sources of HMG1

This example shows which cell sources are capable of releasing HMG1 inresponse to TNF, IL-1 and/or LPS. Cells studied include GH₃ pituicytes,murine macrophage RAW 264.7 cells, human primary peripheral bloodmononuclear cells (huPBMCs), human primary T cells, rat adrenal PC-12cells, and rat primary kidney cells (Table 1). The rat pituitary GH₃cell line was obtained from American Type Culture Collection (ATCC,Rockville, Md., USA), and cultured in DEME supplemented with 10% fetalbovine serum and 1% glutamine. Human PBMCs and T cells were freshlyisolated from whole blood of healthy donors and cultured in RPMI 1640supplemented with 10% human serum as previously described (Zhang et al.,J Exp. Med. 185:1759–1768, 1997). When confluency reached 70–80%, themedium was replaced by serum-free OPTI-MEM I medium and culturesstimulated with proinflammatory cytokines (e.g., TNFα or IL-1) orbacterial endotoxin (LPS).

Although human T cell, rat adrenal (PC-12) cells, and rat primary kidneycells contained cell-associated HMG1 as demonstrated by Western blottinganalysis of whole cell lysates using HMG1-specific antibodies (seeExample 4 below), HMG1 did not significantly accumulate in the medium ofthese cultures after stimulation with either TNF, IL-1β, or LPS (Table1).

TABLE 1 Induced release of HMG1 from various types of cells. StimulusCell Type TNF IL-1β LPS Murine RAW 264.7 cells Yes Yes Yes Human PBMCsYes Yes Yes Human primary T cells No No No Rat adrenal PC-12 cells No NoNo Rat pituitary GH₃ cells Yes Yes No Rat primary kidney cells No No NoNote: PBMCs, peripheral blood mononuclear cells.TNF, IL-1β (minimal effective concentration=5 ng/ml for each) andbacterial endotoxin (LPS, minimal effective concentration=10 ng/ml)induced the release of HMG1 from human PBMCs in a time- anddose-dependent manner (Table 1). IFN-γ alone (0–200 U/ml) did not induceHMG1 release from any of the above cells, but when added in combinationeither with TNF or IL-1β, IFN-γ dose-dependently enhanced HMG1 releasefrom macrophages, with a maximal 3-fold enhancement by IFN-γ at aconcentration of 100 U/ml. The release of HMG 1 was not due to celldeath, because cell viability was unaffected by TNF, IL-1β, or LPS, asjudged by trypan blue exclusion (90–92±5% viable for control vs.88–95±4% in the presence of 100 ng/ml TNF, IL-1β or LPS). The amount ofHMG1 released by pituicytes and macrophages inversely correlated withthe intracellular concentration of HMG1, as determined by Westernblotting analysis, indicating that the released material is, in partderived from pre-formed cell-associated HMG1 protein.

Potential sources of circulating HMG1 in vivo were assessed byhybridization of an HMG1-specific probe to mRNA prepared from variousnormal human tissues (blot substrate available from commercial sources),with the results summarized in FIG. 5. Several macrophage-rich tissues(lung, liver, kidney, pancreas and spleen) exhibited the most abundantHMG1 mRNA expression; less was observed in pituitary, bone marrow,thymus, lymph node and adrenal gland. In addition to providinginformation as to the relative tissue distribution of HMG1 expression,this study shows the practicality and utility of assaying forHMG1-specific nucleic acid sequences in tissue samples.

EXAMPLE 3 Recombinant HMG1 Administration, in vitro and in vivo

This example details procedures to produce HMG1 by well-knownrecombinant DNA technologies. The HMG1 open reading frame was amplifiedby PCR and subcloned into an expression vector (pCAL-n). Briefly, the648-bp open reading frame of HMG1 cDNA was PCR amplified (94° C. 1′, 56°C. 2′, 72° C. 45″, 30 cycles) from 5 ng Rat Brain Quick-Clone cDNA(Catalog #7150-1, Clontech, Palo Alto, Calif., USA) using primerscontaining the following sequences, 5′-CCC GCG GAT CCA TCG AGG GAA GGATGG GCA AAG GAG ATC CTA-3′[SEQ ID NO. 2], and 5′-CCC GCA AGC TTA TTC ATCATC ATC ATC TTC T-3′[SEQ ID NO. 3]. The 680 bp PCR product (4 μg) wasdigested with Bam HI and Hind III, and cloned into the Bam HI/Hind IIIcloning sites of the pCAL-n vector (Stratagene, La Jolla, Calif., USA).The recombinant plasmid was transformed into E. coli BL21(DE3)pLysS(Novagen, Madison, Wis., USA), and positive clones were screened andconfirmed by DNA sequencing on both strands using a Tag DyeDeoxyterminator cycle sequencing kit on the ABI 373A automated fluorescentsequencer (Applied Biosystems, Foster City, Calif., USA).

To express recombinant HMG1, positive clones were cultured at 37° C.with vigorous shaking (250 rpm) until OD₆₀₀ reached 0.6, when IPTG (1mM) was added. Twelve hours after IPTG induction, bacterial cells wereharvested by centrifugation (6500 rpm, 15 minutes), and lysed byfreeze-thaw cycles. The water-soluble fraction was collected aftercentrifugation (30 minutes, 12,000 rpm), and recombinant HMG1 waspurified on a calmodulin-binding resin column as instructed by themanufacturer (Stratagene). Bacterial endotoxin was removed from therecombinant HMG1 by using Detoxi-Gel endotoxin-removing gel (Pierce,Rockford, Ill. USA, Cat. #20344), and residual LPS content wasdetermined by the Limulus Amebocyte Lysate Test (LAL, test, Cat.#50-648U, QCL-1000 Chromogenic LAL, Bio-Whittaker, Inc., Walkersville,Md., USA). Purified recombinant HMG1 was added to cultures of humanperipheral blood mononuclear cells (HuPBMCs), and supernatants assayedfor TNF by ELISA four hours after stimulation. The LPS-neutralizingagent polymyxin B (10 μg/ml) was added concurrently with recombinantHMG1 to eliminate the effect of any contaminating LPS on TNF release.Additionally, recombinantly derived HMG1 was administered to testanimals, with or without the additional endotoxemic challenge ofexogenous LPS, to study the pathogenic potential of high levels of HMG1in vivo (see FIGS. 2B and 2C). In some experiments, serum samples weresecured from HMG1-treated animals to be assayed for TNF as detailedherein (see FIG. 1B).

The above procedure provides recombinant HMG1 as a fusion peptidecomprising a 3.0 kDa calmodulin-binding domain and a thrombin cleavagesite as an amino terminal extension in register with the HMG1 peptidesequence. In some experiments, the fusion tag was removed from analiquot of the recombinant protein and the bioactivity of the fullfusion protein was compared to the cleaved HMG1 peptide; no significantdifference in bioactivity was noted and additional experiments(especially those requiring administration of recombinantly producedHMG1 to animals) typically were conducted with the (uncleaved) fusionprotein.

As demonstrated in FIGS. 3A and 3B, in vitro or in vivo administrationof recombinantly derived HMG1 induced a brisk TNF response, confirmingthe identification of HMG1 as a late-appearing LPS-inducedmacrophage-derived endogenous mediator with pro-inflammatory activity.

EXAMPLE 4 Anti-HMG1 Antibodies and Immunodetection

This example provides the results of experiments to generate and usepolyclonal antibodies against HMG1. Briefly, polyclonal antibodiesagainst an oligopeptide corresponding to the N-terminal amino acidsequence of HMG1, or against purified recombinant HMG1, were generatedin rabbits according to standard procedures well known in the art.Briefly, eight copies of an oligopeptide with the sequenceGKGDPKKPRGKMSSC [SEQ ED NO. 4] were anchored to radially branchinglysine dendrites (small immunogenically inert core). These largemacromolecules were injected three times both subcutaneously andintradermally (0.5–1.0 mg per injection) into rabbits at week 1, 2, and4 after pre-bleed at Day 0. Two weeks after the last immunization,rabbits were bled and boosted intramuscularly with 1.0 mg of antigen,followed by a second bleeding two weeks later. Alternatively, to producepolyclonal antibodies against recombinant HMG1, rabbits were immunizedwith recombinant HMG1 fusion peptide (100 μg per injection) following asimilar protocol. Monoclonal antibodies reactive against HMG1 (i.e.,that bind, and in some cases, neutralize or antagonize the biologicalactivity of HMG1) are conveniently prepared according to methods wellknown in the art using the HMG1 antigens described herein or other HMG1peptide fragments as immunogens. Such monoclonal antibodies, and/or thehybridomas that produce them, are useful to produce various “humanized”antibodies reactive against HMG1 (all according to methods known in theart), which humanized antibodies; are useful as taught herein.

HMG1-specific antibodies were used to measure by Western blottinganalysis the inducible release of HMG1 from RAW 264.7 cells aftertreatment with TNF or LPS (FIG. 1). Briefly, proteins were fractionatedby SDS-PAGE on a 4–20% gradient gel, transferred to a PVDF membrane, andblotted with rabbit antiserum raised against either the N-terminalsynthetic HMG1 antigen or against recombinant HMG1. The signal wasdetected using a ECL kit as instructed by the manufacturer (AmershamLife Science Inc., Arlington Heights, Ill., USA), and levels of HMG1were determined by measuring optical intensity of bands on Western blotsdigitized for analysis using NIH 1.59 image software, with reference toa standard curve of purified recombinant HMG1.

No HMG1 protein was detected in RAW 264.7 cells-conditioned medium inthe absence, of TNF or LPS treatment, but HMG1 accumulated inconditioned medium to high levels after such stimulation, reaching aplateau at 8–28 hours after stimulation (FIG. 1A). In summary, the datapresented in Examples 1, 3 and in FIG. 1A show that the release of HMG1from macrophages is stimulus-specific and time- and dose-dependent, withmaximal accumulation observed within 8 hours after stimulation with TNFat concentrations as low as 5 ng/ml. It is well appreciated that sepsis,septic shock and related conditions may occur in humans in response tostimuli that differ qualitatively or quantitatively from the singlelarge, lethal LPS bolus used in this predictive models. Nevertheless,experimental endotoxemia has been a valuable and predictive model systemby which to identify critical components of the inflammatory cytokinecascade and by which to identify specific antagonists with predictedclinical utility. In this regard, HMG1 antagonists are perhaps moretherapeutically attractive than TNF antagonists in view of the laterappearance of HMG1 versus TNF in the response to endotoxin.

EXAMPLE 5 Detection of HMG1 in in vivo Animal Models

This example illustrates an in vivo experiment in rodents measuringserum HMG1 levels after administration of a sublethal dose of LPS(LD₅₀). Mice or rats were treated with LPS, and sera were collected atdifferent time points, and assayed for levels of HMG1 by Westernblotting analysis. The serum concentrations of HMG1 were estimated bymeasuring the optical band intensity with reference to a standard curveof purified HMG1. Serum levels increased significantly by 16 hours afterLPS, and remained high for at least 32 hours (FIG. 1B), and were notdetectable in vehicle-treated control animals. These data show that HMG1represents a particularly attractive target for diagnosis of, andpharmaceutical intervention against, sepsis and related disorders ofcytokine toxicity because HMG1 is a late-appearing mediator in theinflammatory cytokine cascade.

EXAMPLE 6 Benefits of Protection Against HMG1

This example provides the results of a predictive in vivo assay tomeasure therapeutic activity of antagonists of HMG1 in relation totreatment of sepsis and related conditions of cytokine-mediatedtoxicity. In this example, the HMG1 antagonist was an anti-HMG1 antibodypreparation. Controls treated with pre-immune serum developed lethargy,piloerection, diarrhea, and succumbed to death within 48 hours. Theseclinical signs of endotoxemia were significantly prevented byadministration of anti-HMG1 antibodies. Male Balb/C mice (6–7 weeks,20–23 grams) were randomly grouped (10 animals per group) andpre-treated either with control (pre-immune) or anti-HMG1 serum (as madein Example 4) 30 minutes before administration (intraperitoneally) of alethal dose of LPS (50 mg/kg in 1×PBS). Other experimental groupsreceived additional doses of anti-HMG1 serum at +12 or, +12, and +36hours after LPS administration. Animals were observed for appearance andsurvival for at least two weeks.

Polyclonal antibodies against recombinant HMG1 were generated inrabbits, and antiserum was assayed for specificity and titer by ELISAand Western blotting procedures. The polyclonal antiserumimmunospecifically recognized (bound to) recombinant HMG1 in Westernblot analysis, for instance, and discriminated rHMG1 from other proteinsin both crude bacterial lysates and as a purified protein that had beendiluted into mouse serum. Using chemiluminescence-amplified detectionmethods in Western blotting analysis, polyclonal anti-HMG1 antiserum atdilutions up to 1:1000 was useful to detect as little as 50 pg rHMG1protein. Administration of anti-HMG1 antiserum in the indicated (FIG.2A) amounts at −0.5 (if one dose), −0.5 and 12 (if two doses), or −0.5,12 and 36 (if three doses) hours relative to LPS challenge (at time 0)was protective against LPS-induced lethality, and repeated dosingschedules provided better protection.

FIG. 2B illustrates that rHMG1 causes dose-dependent lethality inendotoxic mice. Male Balb/C mice (20–23 grams) were randomized in groupsoften to receive LPS (3.15 mg/kg; a non-lethal dose) alone or incombination with purified recombinant HMG1 protein. Administration ofHMG1 at the indicated doses 2, 16, 28 and 40 hours after LPS challengesignificantly increased the lethality of the underlying endotoxemia

FIG. 2C illustrates the independent lethal toxicity of HMG1 as afunction of dose. Purified rHMG1 was administered to male Balb/C mice(five mice per treatment group) as a single i.p. bolus at the indicateddosage. Mice were observed for at least 48 hours, and 60% of micetreated with rHMG1 at a dose of 500 μg/mouse died within 24 hours ofrHMG1 challenge, indicating a single dose LD₅₀, of less than 500μg/mouse.

The protection conferred by anti-HMG1 antibodies was specific, becauseadministration of pre-immune serum, which showed no immunospecificreactivity to HMG1 on Western blots, did not spare subjects fromLPS-mediated mortality (FIG. 2A). Moreover, HMG1-specific antibodies didnot cross-react with other macrophage-derived cytokines (e.g. IL-1 andTNF), eliminating the possibility that antibodies conferred protectionby binding and thereby neutralizing these mediators. Protection againstsepsis, sepsis associated pathogenesis and sepsis-related diseasesinvolving activation of pro-inflammatory cytokine cascades may beimproved by combination therapy targeted against more than one componentof the cytokine cascade. Antagonists of HMG1 in this regard can becombined with specific antagonists of TNF, IL-1, MIF and otherinflammatory mediators, or with more broadly active antagonists ofinflammatory responses that inhibit multiple components of theinflammatory cascade (e.g., aspirin, NSAIDS, anti-inflammatory steroids,etc.), to provide even more effective therapeutic modalities. Protectionagainst LPS toxicity was antibody dose-related, and more frequent dosingwith higher amounts of antibody reduced mortality by up to 70% (FIG.2A). Mice were observed for at least 2 weeks in all experiments, and nolate mortality occurred, indicating that anti-HMG1 antibody treatmentconfers lasting protection against LPS lethality, and does not merelydelay the time of death.

EXAMPLE 7 HMG1 in Human Disease

This example provides data that establish an association between HMG1and human sepsis, and thereby support an indication for using HMG1antagonists generally and anti-HMG1 antibodies in particular in humansepsis and related conditions of cytokine toxicity. Serum HMG1 levels innormal healthy individuals and critically ill patients were measuredusing the polyclonal antibodies generated as in Example 4 in a Westernblot format with reference to a standard curve of rHMG1. HMG1 was notdetectable in normal controls, but accumulated to high levels incritically patients with sepsis (Table 2).

TABLE 2 Serum appearance of HMG1 in sepsis patients. Patient Age HMG1(#) (year) (ng/ml) Diagnosis Outcome 1 27 <d.l. Normal Healthy 2 34<d.l. Normal Healthy 3 35 <d.l. Normal Healthy 4 36 <d.l. Normal Healthy5 61 <d.l. Normal Healthy 6 31 <d.l. Normal Healthy 7 55 10 Sepsis,anastomotic leak Recovered 8 70  7–20 Sepsis, colonic perforationRecovered 9 44 10–60 Sepsis, MOF, spinal reconstruction Died 10 60 >120Sepsis, MOF, perforated Died gastric ulcer 11 47 >120 Sepsis, MOF,pneumonia Died Note: <d.l.—below detection limit; MOF—Multiple OrganFailure.

These data show that elevated serum HMG1 levels are observed in patientswith sepsis, and the highest levels of serum HMG1 are observed in lethalcases (Table 2). These data further indicate the therapeutic importanceof HMG1 antagonists in sepsis and also provide evidence for thediagnostic utility of an assay for sepsis and severity (i.e., potentiallethality) of sepsis by measuring serum concentrations of HMG1. Thisdiagnostic assay is also useful for diagnosing the severity of alliedconditions involving activation of the inflammatory cytokine cascade.

Additional subjects were screened for serum HMG1 levels in associationwith lethal versus non-lethal sepsis, with results (cumulative withTable 2) as described in FIG. 6. The data summarized in FIG. 6 representserum samples obtained from eight healthy subjects and twenty-fiveseptic patients infected with Gram positive [Bacillus fragilis (1patient), Enterococcus facecalis (1 patient), Streptococcus pneumonia (4patients), Listeria monocytogenes (1 patient), or Staphylococcus aureus(2 patients)], Gram negative [Escherichia coli (7 patients), Klebsiellapneumonia (1 patient), Acinetobacter calcoaceticus (1 patient),Pseudomonas aeruginosa (1 patient), Fusobacterium nucleatum (1 patient),Citrobacter freundii (1 patient)], or unidentified pathogens (5patients). Serum was fractionated by SDS-PAGE gel electrophoresis, andHMG1 levels were determined by Western blotting analysis with referenceto standard curves of purified rHMG1 diluted in normal human serum. Thedetection limit by Western blotting analysis is 50 pg. Note that HMG1 isnot detectable in normal controls, but significantly increased in septicpatients. The average level of HMG1 in serum of non-surviving septicpatients (N=13 patients, mean HMG1 level=83.7±22.3 ng/ml) issignificantly higher than in survivors (N=12, mean HMG1 level-25.2±15.1ng/ml, P<0.05). These data provide direct evidence of the utility ofscreening tissue (including, without limitation blood or serum) samplesfor HMG1 sequences (protein or nucleic acid) as a diagnostic andprognostic indicator of the presence of sepsis and related disorders ofcytokine activation and of the severity and likely clinical course ofsuch diseases and conditions.

EXAMPLE 8 HMG1 Induces Pro-Inflammatory Mediators and Weight Loss

The present results provide evidence that HMG1 is a late releasedmediator element of the inflammatory cytokine cascade. Addition ofrecombinant HMG1 to primary human peripheral blood mononuclear cells ledto the dose-dependent induction of TNF within four hours afterstimulation (FIG. 3A). This stimulation by recombinant HMG1 of TNFrelease by HuPBMCs was not due to LPS contamination because: i) purifiedrecombinant HMG1 was not contaminated by LPS as judged by an LALendotoxin assay; ii) addition of the LPS-neutralizing agent polymyxin Bdid not affect HMG1-induced TNF release; and iii) proteolytic cleavageof recombinant HMG1 preparations with trypsin completely abolished theTNF release activity for the PBMC cultures. HMG1 stimulation alsoinduced macrophages to release nitric oxide (NO).

To confirm that HMG1 induced serum TNF release it vivo, purifiedrecombinant HMG1 was administered intraperitoneally to Balb/C mice, andblood samples were collected to be assayed for TNF by the L929 assay. Asshown in FIG. 3B, TNF was not detectable in serum of control animals,but was significantly increased two hours after administration ofrecombinant HMG1 protein.

Repetitive administration of recombinant gene product of the HMG1 gene(100 μg/mouse/day) caused significant body weight loss (FIG. 4) in mice.Without limitation as to mechanism and without being bound by theory,these data are consistent with the hypothesis that HMG1 acts as afeed-forward stimulator of the pro-inflammatory cascade under both invitro and in vivo conditions. These in vivo data in a predictive modelof weight loss also provide predictive evidence that a pharmaceuticalformulation comprising HMG1, or a therapeutically active fragmentthereof, is an effective weight loss therapy.

EXAMPLE 9 In vivo Sources of HMG1

Serum HMG1 levels in hypophysectomized versus control rats also weremeasured by quantitation of Western blot intensities as described above.There were significantly higher HMG 1 levels within 12 hours afterendotoxic challenge (LPS at 1.0 mg/kg) in hypophysectomizid rats(approx. 75 ng/ml) as compared to controls (approx. 25 ng/ml). Theseresults indicate that pituicytes are not the major source of serum HMG1levels and that macrophages may play a quantitatively more importantrole.

EXAMPLE 10 HMG1 A Box Polypeptide Antagonizes HMG1 Induced CytokineActivity in a Dose Dependent Manner

An HMG1 fragment, termed the “A box” (a polypeptide fragment of HMG1having the amino acid sequence: pdasvnfsef skkcserwkt msakekgkfedmakadkary eremktyipp kget (SEQ ID NO: 6)), does not stimulate TNFactivity the extent that the full-length HMG1 protein does. Weakagonists are by definition antagonists; and therefore, the ability ofHMG1 A box to act as an antagonist of HMG1 activity was evaluated. Thisstudy was carried out as follows. Sub-confluent RAW 264.7 cells in24-well dishes were treated with HMG1 (1 μg/ml) and 0, 5, 10, or 25μg/ml of A box for 16 hours in Opti-MEM I medium in the presence ofpolymyxin B (100 units/ml). The TNF-stimulating activity (assayed usingan L929 cytotoxicity assay described by Bianchi et al., Journal ofExperimental Medicine 183: 927–936, 1996) in the sample receiving no Abox was expressed as 100%, and the inhibition by A box was expressed aspercent of HMG1 alone. The results of the effect of A box on TNF releasefrom RAW 264.7 cells is shown in FIG. 7. As shown in FIG. 7, the A boxdose-dependently inhibited HMG1 induced TNF release with an apparentEC₅₀ of approximately 7.5 μg/ml. Data in FIG. 7 are presented as mean SD(n=2–3 independent experiments).

EXAMPLE 11 HMG1 A Box Polypeptide Inhibits Full Length HMG1 CytokineActivity

Antagonism of full length HMG1 activity by an HMG1 A box polypeptide ora GST tag (vector control) was also determined by measuring TNF releasefrom RAW 264.7 macrophage cultures stimulated by co-addition of HMG1 Abox polypeptide with full length HMG1. RAW 264.7 macrophage cells (ATCC)were seeded into 24-well tissue culture plates and used at 90%confluence. The cells were treated with HMG1 alone, or in combinationwith A box polypeptide for 16 hours in Optimum I medium (LifeTechnologies, Grand Island, N.Y.) in the presence of polymyxin B (100units/ml, Sigma, St. Louis, Mo.) and supernatants were collected for TNFmeasurement (mouse ELISA kit from R&D System Inc, Minneapolis, Minn.).TNF-inducing activity was expressed as a percentage of the activityachieved with HMG-1 alone. The results of these studies are shown inFIG. 8. FIG. 8 is a histogram of the effect of HMG1 alone, A boxpolypeptide alone, Vector (control) alone, HMG1 in combination with Abox polypeptide, and HMG1 in combination with vector. As shown in FIG.8, HMG1 A box polypeptide significantly attenuated the TNF stimulatingactivity of full length HMG1.

1. A method for treating a cardiovascular condition characterized byactivation of the inflammatory cytokine cascade, comprisingadministering an effective amount of an antibody that specifically bindsan HMG1 protein or an antigenic fragment thereof, wherein said antibodyinhibits HMG1-mediated activation of the inflammatory cytokine cascade.2. The method of claim 1, wherein said antibody is a polyclonalantibody.
 3. The method of claim 1, wherein said antibody is amonoclonal antibody.
 4. The method of claim 1, wherein said antibody isa chimeric antibody.
 5. The method of claim 1, wherein said antibody isa single chain antibody.
 6. The method of claim 1, wherein said antibodyis a human antibody.
 7. The method of claim 1, wherein said antibody isa humanized antibody.
 8. The method of claim 1, wherein said antibodycan bind to a peptide consisting of the amino acid sequenceGKGDPKKPRGKMSSC (SEQ ID NO:4).
 9. A method for treating a cardiovascularcondition characterized by activation of the inflammatory cytokinecascade, comprising administering a pharmaceutical compositioncomprising an effective amount of an antibody that specifically binds anHMG1 protein or an antigenic fragment thereof, wherein said antibodyinhibits HMG1-mediated activation of the inflammatory cytokine cascade.10. A method for treating a cardiovascular condition characterized byactivation of the inflammatory cytokine cascade, comprisingadministering an effective amount of an antibody fragment thatspecifically binds an HMG1 protein or an antigenic fragment thereof,wherein said antibody fragment inhibits HMG1-mediated activation of theinflammatory cytokine cascade.
 11. The method of claim 10, wherein saidantibody fragment is an antibody fragment of a polyclonal antibody. 12.The method of claim 10, wherein said antibody fragment is an antibodyfragment of a mono clonal antibody.
 13. The method of claim 10, whereinsaid antibody fragment is an antibody fragment of a chimeric antibody.14. The method of claim 10, wherein said antibody fragment is anantibody fragment of a single chain antibody.
 15. The method of claim10, wherein said antibody fragment is an antibody fragment of a humanantibody.
 16. The method of claim 10, wherein said antibody fragment isan antibody fragment of a humanized antibody.
 17. The method of claim10, wherein said antibody fragment is an Fab fragment.
 18. The method ofclaim 10, wherein said antibody fragment can bind to a peptideconsisting of the amino acid sequence GKGDPKKPRGKMSSC (SEQ ID NO:4). 19.A method for treating a cardiovascular condition characterized byactivation of the inflammatory cytokine cascade, comprisingadministering a pharmaceutical composition comprising an effectiveamount of an antibody fragment that specifically binds an HMG1 proteinor an antigenic fragment thereof, wherein said antibody fragmentinhibits HMG1-mediated activation of the inflammatory cytokine cascade.